1. Field of the Invention
The present invention relates to the identification of new muteins of fibroblast growth factor 21 that have reduced O-linked glycosylation when expressed in yeast.
2. Description of the Related Art
Fibroblast growth factors are large polypeptides widely expressed in developing and adult tissues (Baird et al., Cancer Cells, 3:239-243, 1991) and play crucial roles in multiple physiological functions including angiogenesis, mitogenesis, pattern formation, cellular differentiation, metabolic regulation and repair of tissue injury (McKeehan et al., Prog. Nucleic Acid Res. Mol. Biol. 59:135-176, 1998). According to the published literature, the FGF family now consists of at least twenty-three members, FGF-1 to FGF-23 (Reuss et al., Cell Tissue Res. 313:139-157 (2003).
Fibroblast growth factor-21 (FGF-21) has been reported to be preferentially expressed in the liver (Nishimura et al., Biochimica et Biophysica Acta, 1492:203-206, 2000); WO01/36640; and WO01/18172) and described as a treatment for ischemic vascular disease, wound healing, and diseases associated with loss of pulmonary, bronchia or alveolar cell function and numerous other disorders. More recently, FGF-21 has been shown to stimulate glucose-uptake in mouse 3T3-L1 adipocytes after prolonged treatment (72 h), in the presence and absence of insulin, and to decrease fed and fasting blood glucose, triglycerides, and glucagon levels in ob/ob and db/db mice and 8 week old ZDF rats in a dose-dependant manner, thus, providing the basis for the use of FGF-21 as a therapy for treating diabetes and obesity (WO03/011213).
The development of recombinant DNA technology has made possible the production of foreign products such as muteins of FGF-21 in host cells in which exogenous DNA sequences coding for those products have been introduced. The advantage of this technology is that products can be produced in high yields, in highly purified form, with low risk of contamination such as viral contamination. These recombinant techniques have been widely used for the production of recombinant proteins in prokaryotic as well as eukaryotic host cells.
However, the large-scale production of recombinant products by these techniques is still limited, due to problems of expression efficiency of these exogenous DNA sequences, due also to vector instability and to intracellular degradation of the recombinant products by the host cell in which they are made. In addition, recombinant products are often different from their natural counterparts. For example, recombinant products produced in heterologous eukaryotic hosts usually differ from their naturally-occurring counterpart in their glycosylation content. This may concern the presence versus absence of any carbohydrate structure, the localization of said carbohydrate structure on the product, as well as the nature of the carbohydrate. More specifically, it has been shown that yeast-derived recombinant products often bear additional unnatural O-glycans compared to their natural counterpart (Van den Steen, et al., Crit. Reviews in Biochem. and Mole. Biol. 33(3): 151-208, 1998).
The present invention solves the problem of abnormal O-glycosylation associated with yeast-derived recombinant proteins by providing FGF-21 muteins that have a reduced amount for O-glycosylation compared to wild type FGF-21 when expressed in yeast. Applicants have found that the FGF-21 muteins with reduced O-glycosylation can be produced in industrial fermentation conditions and maintain the biological activity necessary to be useful to treat subjects with disorders including, but not limited to, type II diabetes, obesity, and metabolic syndrome.